Host evolution shapes gut microbiome composition in Astyanax mexicanus

Abstract The ecological and genetic changes that underlie the evolution of host–microbe interactions remain elusive, primarily due to challenges in disentangling the variables that alter microbiome composition. To understand the impact of host habitat, host genetics, and evolutionary history on microbial community structure, we examined gut microbiomes of river‐ and three cave‐adapted morphotypes of the Mexican tetra, Astyanax mexicanus, in their natural environments and under controlled laboratory conditions. Field‐collected samples were dominated by very few taxa and showed considerable interindividual variation. We found that lab‐reared fish exhibited increased microbiome richness and distinct composition compared to their wild counterparts, underscoring the significant influence of habitat. Most notably, however, we found that morphotypes reared on the same diet throughout life developed distinct microbiomes suggesting that genetic loci resulting from cavefish evolution shape microbiome composition. We observed stable differences in Fusobacteriota abundance between morphotypes and demonstrated that this could be used as a trait for quantitative trait loci mapping to uncover the genetic basis of microbial community structure.

distinct (Brooks et al., 2016).This phenomenon, known as phylosymbiosis, provides evidence that microbiome composition is associated with host evolution (Lim & Bordenstein, 2020).However, the host genetic changes that underlie natural variation in microbiome composition are largely unknown and challenging to investigate in most systems.Such studies require comparing the microbiomes of closely related genetically tractable species or populations that have adapted to known environments.Studying divergent populations of the same species thrust into similar conditions would allow investigation of parallel development of host genetic mechanisms that select microbial communities.
Researchers have bred surface fish and cavefish in laboratories for generations to study the genetic and developmental basis of cavefish traits like reduced aggression (Rodriguez-Morales et al., 2022), increased fat accumulation (Aspiras et al., 2015;Olsen et al., 2023;Xiong et al., 2022), and insulin resistance (Riddle, Aspiras, et al., 2018).These traits have been linked with microbiome composition in other species (Jia et al., 2021;Qin et al., 2012;Turnbaugh et al., 2006).Characterizing the microbiome of A. mexicanus therefore represents an important step in understanding cavefish evolution.A previous study that compared the stomach microbiome of two field-collected Pachón cavefish and four field-collected surface fish suggested that dissolved oxygen, more so than morphotype identity, determined microbial community composition (Ornelas-García et al., 2018).Here, we characterize the intestinal microbiome of cavefish and surface fish that were collected in the field or raised in the laboratory to ask: (1) Do cavefish and surface fish in their natural habitat have different microbiomes?(2) How similar are the microbiomes of wild and laboratory-raised A. mexicanus?(3) Does host evolutionary history shape microbiome composition in A. mexicanus?
As anticipated, we found that the microbiome is impacted by habitat.Our data shows that laboratory-raised fish harbor a more diverse microbiome that is significantly different in composition compared to their wild counterparts.More surprisingly, we found that host genetics alone can explain differences in microbiome composition between surface fish and cavefish.In addition, we found stable differences in the abundance of Fusobacteriota between laboratoryraised populations suggesting that taxa abundance could be used as a quantitative trait for genetic mapping in A. mexicanus as has been done in mice (Kemis et al., 2019;Zhang et al., 2023).Our study defines the variables that shape the microbiome in a model system that will continue to impact our understanding of ecology and evolution due to its unique phylogeny (Herman et al., 2018;Ornelas-García & Pedraza-Lara, 2015), amenability to laboratory manipulation (Peuß  et al., 2019;Riddle, Martineau, et al., 2018), and genetic accessibility (Klaassen et al., 2018;Stahl, Peuß, et al., 2019).

| Collection of intestinal contents from wild fish
The Pachón Cave is perpetually dark, and previous studies analyzing gut contents of field-caught fish indicate that adults are opportunistic generalists with a seasonally variable diet of mostly detritus and bat guano (Espinasa et al., 2017;Wilson et al., 2021).In contrast, surface fish have a normal day/night cycle and consume insects, crustaceans, and annelids (Mills, 1989)  The weight of the fish used for sequencing is shown in Figure S1a.

| Collection of intestinal contents from laboratory-reared fish
Total intestinal contents were collected from Río Choy surface fish and Tinaja, Molino, and Pachón cavefish that were raised on the same recirculating water system and diet as described below.These fish originated from parents that were bred for at least five generations in the laboratory of Clifford Tabin at Harvard Medical School (TMR2, SMR1, PMR1, MMR7).To minimize the transfer of parental microbes to the embryos, we removed them from spawning tanks and incubated them in 0.3096% sodium hypochlorite (bleach) for 5 min, then 3.2 mM Sodium thiosulfate for 1 min, then rinsed them in fish-ready water (adapted from Dahm & Nüsslein-Volhard, 2002).
The embryos were allowed to hatch in 1 L containers at a density of 20 fish per container.The fish were fed L-type rotifers to 14 days post fertilization, then transferred to 20-gallon tanks on the same recirculating system at a density of approximately 2 fish per liter of water.The fish were fed Artemia fransiscana until 60 days post fertilization, then New Life Spectrum Thera-A Medium Sinking Pellets.
At 6-months post fertilization, fish were euthanized in MS-222 5 h after eating.The fish were weighed, and total intestinal contents were collected using the same method used for field-caught fish.
Samples were stored at room temperature for 24 h, then stored at −20°C until DNA extraction was carried out.The sex of most surface fish samples was apparent while most cavefish samples did not have clearly visible gonads.Gut contents from nine surface (three male, four female, two undifferentiated), nine Pachón (one male, eight undifferentiated), eight Tinaja (eight undifferentiated), nine Molino (six male, three undifferentiated) were shipped on dry ice to Microbiome Insights Inc. (Vancouver, BC, Canada) for DNA isolation and sequencing.The weight and sex of fish used for sequencing is reported in Figure S1b and Table S2.

| DNA extraction and 16S rRNA gene sequencing
Total intestinal contents from each fish were placed into a MoBio PowerMag Soil DNA Isolation Bead Plate.DNA was extracted following MoBio's instructions on a KingFisher robot.Bacterial 16S rRNA genes were PCR-amplified with dual-barcoded primers targeting the V4 region (515F 5′-GTGCCAGCMGCCGCGGTAA-3′, and 806R 5′-GGACTACHVGGGTWTCTAAT-3′), as per the protocol of Kozich et al. (2013).Amplicons were sequenced with an Illumina MiSeq using the 300-bp paired-end kit (v.3), with all samples of this study being included in the same run.

| Data processing for microbiome analysis
Fastq files were imported and processed by Qiime2 (v 2021-11) (Bolyen et al., 2019) [https:// qiime2.org/ ] following the public tutorial for Casava 1.8 paired end demultiplexed sample format.Sequences were denoised using the DADA2 method (Callahan et al., 2016) via qiime data2 denoise-paired to obtain an amplicon sequencing variant (ASV) table.Taxonomy was assigned to ASVs by qiime feature-classifier (Bokulich et al., 2018) (classify-sklearn) against the pre-trained Silva v138 database (Quast et al., 2013) or V4 region (515F/806R) using RESCRIPt (Robeson et al., 2021).Untargeted sequences were removed.The potential for contamination was addressed by co-sequencing DNA amplified from specimens and from two each of template-free controls and extraction kit reagents processed the same way as the specimens.Control samples contained less than 34 read counts.Experimental samples with less than 1000 high-quality read counts were excluded from downstream analysis.
An average of 8197 quality-filtered reads were generated per wild sample.An average of 23,408 quality-filtered reads were generated per laboratory sample.The total number of ASVs from the wild and laboratory datasets was 4555 (including those occurring once with a count of 1, or singletons).

| Quantification and statistical analysis
Alpha diversity was estimated by different indices including Shannon, Inverse Simpson, Observed Species, and Phylogenetic Index on raw count ASV table after filtering out contaminants using the Phyloseq R package (McMurdie & Holmes, 2013).The significance of diversity differences was tested with one-way ANOVA with Tukey's post hoc test including morphotype and habitat as variables (Indices~morphotype*habitat).To estimate beta diversity across samples, we computed Bray-Curtis, Unweighted Unifrac (Lozupone et al., 2011), and Jaccard distance using the vegan v2.6-2 (Oksanen et al., 2015) and Phyloseq R package.We visualized differences across samples using Principal Coordinate Analysis (PCoA) ordination.Variation in microbial diversity was assessed with permutational multivariate analyses of variance (adonis in R) with population and location as factors using 9999 permutations for significance testing.To generate the microbiota dendrogram we collapsed raw ASV table counts from laboratory samples by host morphotype identity to get representative microbiota profiles using the microbiome R package (Lahti & Shetty, 2017).We calculated unweighted unifrac distances for each table and the matrices were Unweighted Pair Group Method with Arithmetic mean (UPGMA) clustered to generate dendrograms of interspecific relatedness using phangorn v 2.9.0 (Schliep, 2011) and ggtree (Yu, 2020) packages.All analyses were conducted in the R environment (R-Core-Team, 2019).Data were plotted using the ggplot2 R package (Wickham, 2016).

| Quantitative PCR of Fusobacteriota abundance
Siblings of the surface fish and Pachón cavefish used for microbiome sequencing (SMR1, PMR1) were shipped from Harvard Medical School to University of Nevada, Reno, in March 2021 when they were 20 months old.Fish were housed in 9 L tanks with 10 fish per tank under 6500 K daylight LEDs and fed ⅛ tsp of New Life Spectrum Thera+A Regular Pellet Enhanced Non-Medicated Fish Food per day for 4 months before fecal collections.To collect feces, individual fish were moved to 2 L tanks and fed five pellets of New Life Spectra Thera A+ per day.On the third day of isolation, tanks were cleared of any debris, the fish were fed, and the inlet water was turned off.Feces were collected from the tank after 24 h.The water inlet was turned on to recirculate fresh water and the process was repeated over 3 days.Fecal samples were centrifuged, excess water was removed, and they were stored at −80°C until DNA extraction.DNA was extracted from the fecal samples using Zymo Quick-DNA Fecal/Soil Microbe Microprep (D6012).DNA concentration was quantified using an Invitrogen Qubit 4 Fluorometer with the 1× dsDNA High Sensitivity assay kit (Q33230).Quantitative PCR was performed on a BioRad CFX96 Touch Real-Time PCR machine using the following primers to amplify 16s rRNA genes from Fusobacteriota (Fwd: 5′-GGATTTATTGGGCGTAAAGC-3′; Rev: 5′-GGCATTCCTACAAATATCTACGAA-3′) (Boutaga et al., 2005).
We also included reactions to amplify 16s rRNA genes from all Eubacteria to normalize the results to the total amount of bacterial DNA (Fwd 5′-GGTGAATACGTTCCCGG-3′; Rev 5′-TACGGCTACCTTGTTACGACTT-3′) (Kostic et al., 2013).Each reaction contained 1× iTaq™ Universal SYBR® Green Supermix, 100 nM of each primer, and 1.5 ng of DNA.The cycle conditions were 95°C for 5 min, followed by 40 cycles of 95°C for 5 s and 57°C for 30 s. Melt curve analysis was performed at 65-95°C with 5°C increments 5 s/step.Reactions without fecal DNA showed amplification of Eubacterial sequences at high cycle number (>29) consistent with the manufacturing of Taq DNA polymerase (Corless et al., 2000).
Fusobacteriota was not detected in samples without fecal DNA.

| Intestinal microbiome of A. mexicanus cavefish and surface fish in their natural habitats
We first analyzed the microbiome of A. mexicanus surface fish and cavefish morphotypes by comparing fish that were captured in the Rio Choy River and Pachón Cave, respectively.In addition to consuming distinct diets, the fish reside in water with dissimilar chemical qualities; water in the Pachón cave at the time of collection was lower in temperature, dissolved oxygen, conductance, copper, and phosphate (Table S1).Despite these distinct habitats, we did not observe significant differences in the richness or composition of the intestinal microbiome between fish collected at each site (Shannon Index one-way ANOVA with Tukey's post hoc test p.adj = .84,Beta diversity Permanova with pairwise post hoc test p.adj = .098).
We examined the proportional abundance of taxa and found that many fish were dominated by only one or two phyla, and although there was a considerable interindividual variation comparing samples, some phyla that were consistently abundant in surface fish were at low abundance in Pachón cavefish (Figure 2).For example, we observed that Firmicutes were the most abundant phylum in four out of six surface fish and, in contrast, were at relatively low abundance in all four Pachón cavefish samples.Pachón individuals instead were dominated by Proteobacteria, Bacteroidota, or Spirochaetota (Figure 2a,b, average proportional abundance (APA) of Firmicutes = 0.15 Pachón, 0.64 surface, Proteobacteria = 0.62 Pachón, 0.03 surface, Bacteroidota = 0.12 Pachón, 0.09 surface).
Similarly, four out of six surface fish samples were dominated by the genus Clostridium, which was found at very low abundance in only two out of four Pachón cavefish samples (Figure 2c, APA = 0.008 Pachón, 0.52 surface).In addition, two surface fish samples (s3, s4) had a high proportion of the genus Cetobacterium which was found at very low abundance in only one Pachón cavefish (Figure 2c, APA = 0.07 Pachón, 0.20 surface).In contrast, the genus Brevinema was observed at high abundance in one of the Pachón cavefish and was not found in surface fish.Brevinema was first characterized as an infectious pathogen in rodents (Anderson et al., 1987).
In fish, abundance has been associated with high stocking density and upregulation of immune gene expression (Brown et al., 2019;Li et al., 2021;Tapia-Paniagua et al., 2014), suggesting it could represent a pathogen.
We considered that interindividual variation in phyla abundance could be driven by fish weight; although the average weight was not different between populations, four of the field-collected surface fish (S2, S3, S4, S5) were considerably larger than cavefish (Table S2, Figure S1, p = .08,t-test).However, we found that the abundance of Bacteriodota, for example, did not correlate with fish weight (p > .05,Spearman's).Similarly, we did not find a significant effect of fish weight for any of the alpha diversity measures (p-values for Shannon: .1464,InvSimpson: .592,observed diversity .2751,Phylogenetic diversity .1788).Overall, the current data suggests that in their natural habitat, A. mexicanus surface fish and cavefish gut microbiomes are dominated by only a few phyla and that some phyla that are found at high abundance in surface fish are found at low abundance in Pachón cavefish.Since a limited number of samples are available for field collection, it is not possible to control for the age or size of the fish to determine how these variables impact microbiome composition.To address this limitation, we next compared the microbiome of fish bred and reared in the laboratory.

| Comparison of the intestinal microbiome between wild and laboratory-reared A. mexicanus
To understand how similar the microbiome of A. mexicanus in their wild habitat is to those raised in the laboratory, we compared the microbiomes of field-collected surface fish and Pachón cavefish to their laboratory-reared counterparts that had been bred in the laboratory for generations and were raised on the same diet and in the same water.The laboratory habitat is different from both the cave and river habitats.In the laboratory, the fish experience lower temperature and pH, higher conductance and dissolved oxygen, an invariable light cycle, more crowded growth conditions, and a consistent high-nutrient diet (Table S1).We found that laboratory-raised fish had the greater richness of microbial species in the intestinal microbiome compared to fish in the wild habitat (Figure 3a, Shannon Index, Figure S2, total observed species, phylogenetic index); alpha-diversity was significantly different in samples from surface fish and Pachón cavefish collected in the field compared to surface fish and Pachón cavefish raised in the lab (Shannon Diversity Tukey's post-hoc test, wild surface vs. lab surface FDR p.adj = .001,wild Pachón vs. lab Pachón FDR p.adj = .008,Table S3).Our results suggest the laboratory habitat supports the growth of a richer microbial community in the GI tract than in either natural environment.
To obtain a graphical representation of the differences in microbiome composition between the wild and the laboratory-reared fish we summarized ASV abundances into Bray-Curtis dissimilarities and performed principle coordination analysis (PCoA, Figure 3b).In the PCoA plot, wild surface fish and wild Pachón cavefish form two partially overlapping clusters that are separate from laboratory-raised fish.We found that habitat (wild vs. lab) significantly impacted microbiome composition (Table 1, adonis R function, or Permanova, p = .002);beta-diversity was significantly different in samples from surface fish and Pachón cavefish collected in the wild compared to surface fish and Pachón cavefish raised in the lab (Table 2,

| Shared microbial taxa between wild and laboratory-reared A. mexicanus
We next asked if any of the microbes present in the natural habitat have been maintained in the laboratory-reared fish by identifying ASVs that are present in both sample types.We found that 53% of the microbes in the wild surface fish were present in the lab surface fish (72/135 amplicon sequencing variant (ASVs), Figure 3c).
Microbes that are shared between individuals of the same species that have been raised in different habitats have been referred to as the "core microbiome."It is hypothesized that these microbes are essential for the normal function of the host and that host-driven mechanisms may exist to ensure they are represented (Roeselers et al., 2011).By this definition, Pachón cavefish had fewer core microbes compared to surface fish (28 vs. 72 ASVs), and most of the core microbes were also present in the surface fish core microbiome (19 out of the 28 ASVs).The data may suggest that many of the same microbes are essential for host function in surface fish and cavefish, but cavefish could require fewer.Exploring this possibility would require manipulating microbiome composition and studying the impact on the host.The core microbes are mostly from the phylum Firmicutes (Figure S4).Firmicutes are one of the dominant taxa in fish microbiomes and are important for carbohydrate metabolism (Kim et al., 2021).We only identified one microbe that was part of the Pachón core and never found in surface fish, Aeromonas asv.13 (Figure 3c, Figure S4).Aeromonas in fish can exhibit either pathogenic or mutualistic interactions, contingent upon the specific species and host involved (Bates et al., 2006;Li et al., 2020;Rolig et al., 2018;Stephens et al., 2016).Determining whether this "core microbe" reflects a difference in predisposition to infection, rather than a difference in maintenance of microbes that are essential to the host, will require species-level identification and additional experiments.Overall, our results suggest that A. mexicanus in the laboratory has a microbiome that is distinct and more diverse compared to their wild counterparts.In addition, more microbes are shared between lab and wild surface fish compared to lab and wild cavefish.

| Evidence that host identity drives differences in A. mexicanus microbiome composition
We next tested whether the identity of the host population impacts microbiome composition independent of habitat by comparing the intestinal microbiome of 6-month-old fish from the Río Choy River, Pachón Cave, Tinaja Cave, and Molino Cave that have been bred in the laboratory for generations, were surface sterilized as embryos, and were raised on the same recirculating water system and diet.
In this controlled setting, we found that Tinaja and Molino cavefish had significantly lower microbial species richness compared to surface fish suggesting that genetic differences between hosts that shape host traits impact alpha diversity, the number and distribution of microbial species in the intestine (Figure 3a: Shannon Index, Figure S2b: Inverse Simpson Index).
In addition to impacting microbial species richness, we found that morphotype identity had a significant impact on microbiome composition (Table 1, Beta Diversity Permanova, p = .002).To visualize the differences in microbiome composition we summarized ASV abundances into Bray-Curtis dissimilarities and performed principle coordination analysis (PCoA, Figure 3b).In the PCoA plot comparing laboratory raised fish, the centroid of Tinaja cavefish samples is furthest from surface fish samples and the ellipses estimating covariance overlap except for Pachón and Tinaja cavefish samples (Figure 3b).PCoA plots of unweighted unifrac and Jaccard distances differently show that the Pachón centroid is ordinated furthest from surface fish and that ellipses estimating covariances overlap in all samples (Figure S3).To determine which laboratory-reared morphotypes harbored significantly different microbiomes, we performed a pairwise post-hoc test (Table 2).We found that the microbiome of surface fish was significantly different from Pachón and Tinaja cavefish (p.adj = .025and .020)but not Molino cavefish (p.adj = .075).
These results suggest that, through the course of evolution, Tinaja and Pachón cavefish have acquired genetic changes that result in the establishment of a different microbiome from surface fish despite being raised in the same habitat.We also found that samples from Pachón and Tinaja cavefish were significantly different from each other suggesting distinct cavefish populations have genetic differences that impact microbiome composition (p.adj = .006).Since the fish were all raised under identical conditions, these results suggest that genetic and phenotypic differences between A. mexicanus morphotypes drive differences in microbiome composition.

| Differential abundance of microbial taxa in laboratory-reared A. mexicanus
We next compared the microbial taxa between laboratory-raised A. mexicanus morphotypes to gain insight into the potential functional significance of host-driven microbiome differences.We found that the four most abundant phyla were Firmicutes, Proteobacteria, Fusobacteriota, and Bacteroidota like in other freshwater fishes (Kim et al., 2021) (Figure 2a,b, Table S4).However, the average proportional abundance of Firmicutes was highest in surface fish We found that samples from wild Pachón cavefish also had a low proportional abundance of Fusobacteriota compared to samples from wild surface fish (APA = 0.07 Pachón, 0.20 surface).The Fusobacteriota sequences we identified in the A. mexicanus gut microbiome are assigned to the genus Cetobacterium which is common in the microbiome of freshwater fish (Tsuchiya et al., 2007).Our data suggest that genetic differences between A. mexicanus morphotypes alter the taxonomic composition of the intestinal microbiome and that most strikingly, Pachón cavefish have dramatically reduced abundance of Cetobacterium.

| Evidence of phylosymbiosis in A. mexicanus
We next tested if the differences in microbiome composition we observed between laboratory-reared surface fish and cavefish mirror the evolutionary history of A. mexicanus.The A. mexicanus phylogeny defines two lineages; fish from the Pachón and Tinaja caves and Rascón River form a monophyletic clade referred to as the "old lineage" and fish from the Molino Cave and Río Choy River form a monophyletic clade referred to as the "new lineage" (Figure 1h).Based on whole genome sequencing data, old and new lineages split as long as 257 K generations ago with instances of secondary contact (Herman et al., 2018).Molino cavefish split from Río Choy Surface fish around 163 k generations ago, and Pachón and Tinaja cavefish split more recently around 116 k generations ago (Herman et al., 2018).We generated dendrograms of interspecific relatedness using the microbiota profiles of laboratory-reared Pachón, Tinaja, and Molino cavefish and Río Choy surface fish.We found that the topology of the microbiota tree matches the topology of the host phylogeny (Figure 3d).The trees similarly define two lineages; Molino cavefish and Río Choy surface fish form a monophyletic clade that is separate from Pachón and Tinaja cavefish.Our results suggest that phylosymbiosis can occur in the same species that consists of distinct populations that diverged less than 300 K generations ago and adapted to dramatically different habitats.

| Feasibility of genetically mapping microbiome composition in A. mexicanus
Despite their distinct appearance and behavior, A. mexicanus surface and cave morphotypes have remained interfertile.F2 surface/cave hybrids have been used in numerous quantitative trait loci (QTL) mapping studies to identify genetic changes associated with cavefish evolution (Gross et al., 2014;Klaassen et al., 2018;Kowalko, Rohner, Linden, et al., 2013;Protas et al., 2007;Riddle et al., 2021;Warren et al., 2021).Using microbiome composition as a trait for QTL mapping in mice has revealed genomic regions associated with the abundance of bacterial taxa in the intestine (Zhang et al., 2023).
We next investigated the feasibility of using proportional abundance of taxa in the microbiome as a trait for QTL mapping in A. mexicanus by examining how stable bacterial taxa abundance is across time in individual fish.We reasoned that if the trait is highly plastic, it may be less likely to identify genetic markers associated with trait variance at the population level.We focused on Fusobacteriota since proportional abundance of this taxa was the most different between morphotypes.
We found that Fusobacteriota was at very low proportional abundance in 16s rRNA gene sequencing data from gut contents and pooled fecal samples of laboratory-raised Pachón cavefish compared to other morphotypes (Figure 4a,b).Discovering differences in abundance in fecal samples suggested that (1) feces can be used to quantify differences in Fusobacteriota between individuals, and (2) an individual fish could be sampled over time.
We collected feces from individual three-year-old surface fish and Pachón cavefish that were siblings of the 6-month-old fish used for microbiome sequencing.Using quantitative PCR, we found that Fusobacteriota 16s rRNA genes were always detected in the feces of the surface fish (n = 6) and were undetectable (n = 2/6) or had a higher threshold for detection in the feces of most Pachón cavefish (n = 3/4, Figure 4c).Our data suggest that Fusobacteriota abundance varies between individual fish, but that lower abundance in Pachón compared to surface fish is a trait that persists through life.We also found that Fusobacteriota abundance was stable in individual fish sampled at different times; fish with high or low abundance consistently had high or low abundance (Figure 4d).The results demonstrate the feasibility of using proportional abundance of Fusobacteriota as a trait for genetic mapping in A. mexicanus to identify host-driven molecular pathways that shape microbiome composition.We characterized the intestinal microbiome of A. mexicanus surface fish and cavefish morphotypes in their natural habitat and reared under identical conditions in the laboratory to understand (1) if wild cavefish and surface fish have different microbiomes, (2) if the wild microbiomes are represented in the laboratoryreared fish, and (3) if host evolutionary history alone drives differences in microbiome composition.Our results show that in their natural habitat, surface fish and cavefish are dominated by only a few phyla and there is considerable interindividual variation comparing river-dwelling and cave-dwelling fish.We found that the laboratory environment supports the growth of a richer microbial community that contains more of the wild surface fish taxa compared to the wild cavefish taxa.By comparing the microbiomes of surface fish and multiple cavefish morphotypes that were reared in the laboratory, we discovered that host genetics drives differences in microbiome richness and composition and that the microbial community relationships recapitulate the phylogeny of the host.Our study shows that phylosymbiosis can occur within the same species consisting of populations that have adapted to dramatically different habitats.Furthermore, we found consistent and readily quantifiable differences in taxa between A. mexicanus morphotypes that can be used to investigate the genetic basis of microbial community structure and how it impacts the development, physiology, metabolism, and behavior of the host.

| Drivers of microbiome composition in A. mexicanus compared to other fishes
Our study adds to the investigations of what drives microbiome diversity in fishes more broadly.We found that in A. mexicanus, evolutionary history alone can account for differences in gut microbiome composition.Most studies on other fish species have found that habitat is the main driver of microbial diversity.For example, a meta-analysis of 25 fish species revealed that trophic level and salinity were the best predictor of microbiome composition (Sullam et al., 2012).Similarly, a study examining the microbiome of 227 individuals from 85 fish species found that host habitat most strongly shaped the microbiome (Kim et al., 2021).The dominant impact of habitat has also been observed when comparing closely related species; the microbiomes of Cichlids from two lakes cluster by diet (Baldo et al., 2017;Härer et al., 2020).Nevertheless, a role for host phylogeny has been demonstrated in some comparisons.A recent examination of 24 freshwater species from the Yellow River found that diet, location, and host phylogeny predict microbiome composition (Pan et al., 2023).
The studies described above-compared field caught fish and statistically analyzed the impact of habitat on microbiome composition.In contrast, we controlled the variable of habitat by comparing fish that were spawned in the laboratory, consumed the same diet, and were raised in the same water.A study of a similar design using threespine stickleback (Gasterosteus aculeatus) compared the microbiomes of freshwater benthic and freshwater limnetic ecotypes from three different lakes (Rennison et al., 2019).
In contrast to what we observed in A. mexicanus, the ecotypes did not develop different microbiomes when raised in the same conditions.The harsh cave environment may select for traits that have an outsized effect on microbiome composition compared to the variable environments experienced during the adaptive radiation of other fish species.An emerging theme in predicting microbiome composition is that the relative impact of habitat and evolutionary history depends on the ecological forces that shaped the clade being examined.

| Limitations in sampling and methodology
Some studies have shown that age and sex can impact fish microbiome composition (Navarro-Barrón et al., 2019;Piazzon et al., 2019).
Although not significantly different, the field-collected surface fish were larger on average compared to Pachón cavefish and were all sexually differentiated suggesting they may be older (Figure S1).Due to the limited availability of samples, we were not able to test how these variables may impact microbiome composition.However, in the laboratory, we collected fish that were the same age (6 months old).The lab-raised morphotypes were not significantly different in weight at the time of collection, although Pachón and Tinaja cavefish weighed more than surface fish and Molino cavefish on average (Figure S1).Interestingly, most Tinaja and Pachón cavefish had undefined sex compared to surface fish and Molino cavefish (Figure S1).
Additional sampling of fish across different life stages would help delineate the impact of age, size, and sex on microbiome composition in A. mexicanus.
To investigate how host identity impacts microbiome composition, we compared the offspring of surface, Tinaja, Pachón, and Molino A. mexicanus that have been maintained in the same laboratory environment on the same diet for multiple generations.
We removed spawned embryos from parental tanks, treated them with bleach before hatching, and raised them on the same diet.
The morphotypes developed different microbiomes despite experiencing the same environment suggesting host identity is a major driver of microbiome composition.However, we cannot exclude the possibility that bleaching the embryos did not eliminate the influence of the parental microbiome.Vertically transmitted microbes, or microbes that remained associated with the embryos after bleaching could influence the early colonization and subsequent development of the gut microbiota.As stated previously, however, the parents of the fish used in this study were also raised in the laboratory on the same diet.Therefore, if the parental microbiomes differed, it would also be largely driven by the identity of the host.Future studies may utilize germ-free derivation techniques and inoculation with known microbes to understand the strength of the host-driven mechanisms that shape microbiome composition in A. mexicanus.

| Implications for investigating the adaptive benefit of cavefish traits
Our study provides important context for research on the evolution of A. mexicanus behavior and metabolism as these traits are known to be influenced by microbiome composition.For example, cavefish have reduced aggression (Rodriguez-Morales et al., 2022), which can be recapitulated in flies, mice, and hamsters through microbiome manipulation (Gulledge et al., 2023).In addition, cavefish are fat (Xiong et al., 2022) and insulin resistant (Riddle, Aspiras, et al., 2018) which is associated with microbiome changes in fish and mammals (Qin et al., 2012;Turnbaugh et al., 2006).When considering the adaptive benefit of A. mexicanus traits recorded in the laboratory, it is important to note that the laboratory environment does not recapitulate either wild habitat (river or cave) and the fish display considerable plasticity in some traits (Bilandžija et al., 2020).We showed that laboratory-raised fish harbor a more diverse microbiome compared to their field-caught counterparts and that the laboratory microbiome contains more of the taxa in wild surface fish compared to the wild cavefish.Our results serve as a foundation for understanding how different microbial taxa impact A. mexicanus phenotypes which will lead to a better understanding of cavefish evolution.

| Functional significance of differences in taxonomic composition between morphotypes
The taxonomic differences we observed between the microbiomes of laboratory-raised A. mexicanus morphotypes have been associated with host traits in other species.For example, we observed a higher ratio Firmicutes to Bacteroidetes ratio (F/B ratio) in cavefish compared to surface fish (Table S4) which has been linked with obesity in mammals (Stojanov et al., 2020).Cavefish are fatter than surface fish (Xiong et al., 2018) and the role for the microbiome in underlying increased adiposity has yet to be explored.We found that Pachón cavefish samples had a high abundance of Cupriavidus, which is a genus associated with resistance to copper toxicity (Pan et al., 2021).Whether Pachón cavefish are resistant to copper has not been tested but copper concentrations in the Pachón cave were lower than in the surface river at the time of collection (Table S1).
Cupriavidus has been found at higher abundance in human patients with type-2 diabetes compared to healthy individuals (Nah et al., 2019).Interestingly, Pachón cavefish have traits that model diabetes like insulin resistance and higher blood sugar (Riddle, Aspiras, et al., 2018), and a possible connection with the microbiome has not yet been tested.
Another taxonomic difference that shows interesting alignment with Pachón cavefish traits is the near absence of Fusobacteriota of the genus Cetobacterium compared to the other morphotypes.
Cetobacterium is common in the microbiome of herbivorous fish and is thought to assist in carbohydrate metabolism (Tsuchiya et al., 2007).
One predominant species, Cetobacterium somerae, has been shown to produce vitamin B12 which provides resistance to pathogen infection (Qi et al., 2023).C. somerae has also been shown to improve glucose homeostasis when administered to zebrafish, Danio rerio, through the production of acetate and modulation of the gut-brain axis (Wang et al., 2021).Low Cetobacterium abundance in Pachón cavefish could therefore be linked with their observed increased sensitivity to infection (Peuß et al., 2020) and impaired glucose homeostasis (Riddle, Aspiras, et al., 2018).In humans, an overabundance of microbes from the same family (Fusobacteriaceae) play a role in the progression of diseases like periodontitis, appendicitis, and gastrointestinal cancer (Griffen et al., 2012;Kostic et al., 2012;Swidsinski et al., 2011).For example, an abundance of Cetobacterium in combination with other microbes can be used as a diagnostic biomarker for colorectal cancer (Yao et al., 2021).Discovering the genetic basis of low Fusobacteriota abundance in Pachón cavefish may have biomedical relevance in addition to furthering our understanding of cavefish evolution and host-microbe interactions.

| A. mexicanus as a model for investigating host-microbiome interactions
Our study stands apart from other investigations on the factors that influence the fish microbiome due to the nature and future promise of the model system.A. mexicanus consists of multiple cavefish populations of polyphyletic origin allowing investigations of parallel evolution.We found that host-driven microbiome differences exist between and within branches of the A. mexicanus phylogeny providing multiple natural replicates to investigate the evolution of microbiome composition.In most other species it is not possible to compare divergent populations and a representative ancestor in a controlled laboratory setting.A. mexicanus is easy to grow and manipulate in the lab, standardized husbandry protocols are published (Riddle, Martineau, et al., 2018), the genome is sequenced and annotated (Warren et al., 2021), and there are a growing number of resources available to examine and manipulate gene function (Stahl, Jaggard, et al., 2019).Furthermore, methods for deriving germ-free zebrafish, and inoculating them with specific microbes could be applied to A. mexicanus (Melancon et al., 2017).Since post-larval fish are transparent like zebrafish, the biogeography of the A. mexicanus microbiome could be visualized in live fish using fluorescent microbes.Perhaps most importantly, the surface fish and cavefish are interfertile allowing hybridization of surface/cave and cave/cave individuals to investigate the heritability and genetic basis of traits using QTL mapping.
It is possible to generate hundreds of F2 hybrids and use them to evaluate complex interactions between host genotype, phenotype, and microbiome community structure.We showed that proportional abundance of Fusobacteriota is very different between surface fish and Pachón cavefish and stable within individuals suggesting it would be a promising taxon to focus on for future QTL studies.In addition to providing insight into the relative impacts of environment and host in determining microbiome composition, our findings establish A. mexicanus as an evolutionary model to investigate the molecular mechanisms that mediate host-microbe interactions.

| CON CLUS ION
Astyanax mexicanus is a powerful model system to investigate the genetic basis of morphological, physiological, and behavioral evolution due to the ability to compare distinct cavefish morphotypes to their extant surface fish ancestor.We were able to discover and define the effects of environment and host evolutionary history on the gut microbiome of this species due to the ability to study the morphotypes in their natural habitat and compare them under the same controlled conditions in the laboratory.We found that the microbiomes of cavefish and surface fish in the wild are dominated by only a few phyla.Fish in the laboratory have a more diverse microbiome that is distinct in composition compared to their wild counterparts.In addition, there are more shared microbes between lab versus wild surface fish compared to lab versus wild cavefish.Importantly, we determined that host genetics alone can drive differences in microbiome diversity and composition by comparing the microbiomes of surface fish and multiple cavefish morphotypes that were reared in the laboratory under identical conditions.Moreover, our data revealed that the differences in microbiome composition mirror the A. mexicanus phylogeny showing that host evolutionary history within a single species can shape gut microbial community structure.
, appearance and phylogeny of Astyanax mexicanus.(a) Map and sattelite image showing location of A. mexicanus cave morphotype populations used in this study.(b) Image of river in Mexico where A. mexicanus surface fish morphotypes are found.(c) Image of Pachón Cave where A. mexicanus cavefish morphotypes are found.(d-g) Images of surface fish and cavefish (Molino, Pachón, Tinaja) raised in the laboraotry.(h) A. mexicanus phylogeny; Tinaja and Pachón form a monophyletic clade referred to as the "old lineage" (gray shaded area) and Molino and Río Choy surface fish form a monophyletic clade referred to as the "new lineage."Adapted from Ponnimbaduge Perera et al. (2023) with permission.Drawing based on Herman et al. (2018).
cavefish and Río Choy surface fish in July 2019 using the following protocol: Captured fish were placed in their environmental water and euthanized by immersion in MS-222 on the day of capture.The gut was removed by making a cut posterior to the stomach and directly at the urogenital pore.The gut was transferred to a sterile Petri dish and the contents were washed out by inserting a sterile syringe into one end and expelling 200 μL sterile PBS.The contents and PBS were transferred to 300 μL RNA later.The samples were stored at room temperature for 4 days, shipped on ice, and stored at −20°C until DNA extraction was carried out.The PBS and RNA later used to collect samples were included in the DNA extraction and sequencing pipeline and did not produce read counts high enough to analyze.Thirteen out of 20 wild samples that were collected had enough read counts to analyze: five Pachón (three males, one female, one undifferentiated) and six Río Choy (three males, three females).

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I G U R E 2 Taxonomic composition of the intestinal microbiome of wild and laboratory-raised Astyanax mexicanus surface fish and cavefish morphotypes.(a) Proportional abundance by phylum of ASVs identified in wild samples (surface and Pachón) and laboratory raised samples (surface, Pachón, Tinaja, and Molino).(b) Average proportional abundance by phylum of ASVs identified in wild samples and laboratory raised samples.(c) Proportional abundance by genus of ASVs identified in wild samples and laboratory raised samples.(d) Average proportional abundance by genus of ASVs identified in wild samples and laboratory raised samples.
pairwise post-hoc test, wild surface vs. lab surface FDR p.adj = .005,wild Pachón vs. lab Pachón FDR p.adj = .006).In summary, the intestinal microbiome of A. mexicanus in the laboratory has increased richness and altered composition compared to A. mexicanus in their natural habitat.These results have implications for researchers investigating the evolution of cavefish traits that may be influenced by microbiome composition.F I G U R E 3 Comparison of the gut microbiome between wild and laboratory-raised Astyanax mexicanus surface fish and cavefish morphotypes.(a) Comparison of the number of species and their distributions in intestinal microbiome of A. mexicanus surface and cavefish populations as estimated by Shannon Index.Asterisks indicate significance based on one-way ANOVA with Tukey's post hoc test (*p < .05,**p < .005).(b) Variation in intestinal microbiome composition between A. mexicanus surface fish and cavefish in the wild and lab.Distances determined by Bray-Curtis.Ellipses estimate covariance and the centroid of each cluster.PCoA used as the ordination method.(c) Venn diagram showing shared number of ASVs between wild and laboratory-raised surface fish and Pachón cavefish.(d) Dendrogram of interspecific relatedness of the intestinal microbiome of laboratory-raised A. mexicanus.ASV tables were collapsed by the indicated population and Bray-Curtis distances were calculated for each representative microbiota profile.The resulting Bray-Curtis distance matrices were UPGMA clustered to produce the dendrogram.
compared to cavefish which recapitulates what we observed in the natural habitat (Figure 2a,b, APA = 0.40 surface, 0.36 Pachón, 0.21 Tinaja, 0.24 Molino).In addition, the phylum Bacteroidota and genus Bacteroides made up a high proportion of ASVs in surface fish samples compared to cavefish samples (Figure 2c,d, APA = 0.14 surface, 0.10 Pachón, 0.06 Tinaja, 0.07 Molino).We found that Pachón cavefish samples were mostly dominated by Proteobacteria of the genus Cupriavidus which was lower in the other morphotypes (Figure 2c,d, APA = 0.19 Pachón, 0.08 surface, 0.06 Tinaja, 0.09 Molino).The most striking difference in taxonomic composition we observed was a very low abundance of Fusobacteriota in Pachón cavefish compared to the other morphotypes (Figure 2a: dark green bars, Figure 2b: light orange bars).We found that the Fusobacteriota phylum was consistently present in surface fish and was the most abundant phylum in 1 out of 10 surface fish.Proportional abundance of Fusobacteriota was greater in Molino and Tinaja cavefish samples compared to surface fish samples; it was the most common phylum in four out of nine Molino samples and five out of nine Tinaja samples.In contrast, we did not observe it in most Pachón samples (six out of eight) or it was at very low abundance if present (APA = 0.0005 Pachón, 0.08 surface, 0.46 Tinaja, 0.22 Molino).

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Low abundance of Fusobacteriota in Astyanax mexicanus Pachón cavefish can be detected in individual fish over time using fecal samples.(a) Bar graph showing average proportional abundance of Fusobacteriota ASVs in 16s rRNA gene sequencing of intestinal contents of A. mexicanus surface fish and cavefish raised in the laboratory (n = 35).(b) Bar graph showing proportional abundance of Fusobacteriota ASVs in 16s rRNA gene sequencing in pooled fecal samples from A. mexicanus surface fish and cavefish morphotypes raised in the laboratory (n = 4).(c) Quantification of Fusobacteriota 16s rRNA genes and Eubacteria 16s rRNA genes in fecal samples from individual 3-year-old siblings of the surface fish and Pachón cavefish used for intestinal microbiome sequencing.Greater quantification cycle indicates lower abundance.Eubacteria is not different between samples indicating the total amount of bacterial DNA has been properly controlled.(d) Quantification of Fusobacteriota 16s rRNA genes and Eubacteria 16s rRNA genes in fecal samples collected on three different days from individual 3-year-old siblings of surface fish and Pachón cavefish used for microbiome sequencing.

Table S1 .
. Field collection of Astyanax mexicanus for this study was conducted under permit no.SGPA/ DGVS/03634/19 granted by the Secretaría de Medio Ambiente y Recursos Naturales to Ernesto Maldonado.The date and time of collections, and measured water chemistry variables are reported in Total intestinal contents were collected from wild Pachón Statistical comparison of microbiota composition in A. mexicanus gut contents using results of 16s rRNA gene sequencing.